Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes

Day et al. Clinical Epigenetics (2016) 8:77 DOI 10.1186/s13148-016-0246-x RESEARCH Open Access Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes Samantha E. Day1, Richard L. Coletta2, Joon Young Kim3, Latoya E. Campbell1, Tonya R. Benjamin4, Lori R. Roust4, Elena A. De Filippis4, Valentin Dinu5, Gabriel Q. Shaibi6,7, Lawrence J. Mandarino8 and Dawn K. Coletta7,9,10* Abstract Background: Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity. Results: Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 ± 0.7 kg/m2) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m2) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change −1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5'UTR (Chr.8:22,423,530–22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity. Conclusions: These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle. Keywords: Methylation, Next-generation sequencing, Skeletal muscle, Obesity * Correspondence: 7 Mayo/ASU Center for Metabolic and Vascular Biology, Mayo Clinic in Arizona, Scottsdale, AZ, USA 9 School of Nutrition and Health Promotion, College of Health Solutions, Arizona State University, 550 N. 3rd Street, Phoenix, AZ 85004, USA Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Day et al. Clinical Epigenetics (2016) 8:77 Background Obesity is a condition that affects about one third of the US adult population [1]. It is a major disease associated with other co-morbidities, including type 2 diabetes, metabolic syndrome, and cardiovascular disease [2]. An underlying feature of obesity is insulin resistance. Insulin resistance is a reduced biological response of insulin on peripheral tissues including skeletal muscle, liver, and fat [3]. Under normal physiological conditions, skeletal muscle accounts for approximately 80 % of insulinstimulated total body glucose uptake [4]. Previous studies from our laboratory have investigated the molecular mechanisms of insulin resistance in skeletal muscle. We have previously shown that insulin resistance in skeletal muscle is in part due to mitochondrial dysfunction [5]. In experimentally induced insulin resistance, we have shown a low grade inflammatory response, with increases in extracellular matrix (ECM) turnover [6]. Furthermore, by using a proteomic approach on insulin resistant muscle, we identified alterations in the abundance of protein involved in cytoskeletal structure and assembly [7]. Our findings, to date, demonstrate a cross talk relationship between inflammation, extracellular remodeling, cytoskeletal interactions, mitochondrial function, and insulin resistance in human skeletal muscle [8]. The pathogenesis of obesity-associated insulin resistance is due to environmental and genetic factors [9, 10]. However, the role of epigenetic factors, which may provide a potential link between the genetic and environmental factors observed in obesity, is poorly understood. Epigenetics can be described as heritable changes in gene function that occur without a change in nucleotide sequence [11]. DNA methylation is an epigenetic modification and is generally observed as a methyl addition to the carbon 5 position of cytosines and more commonly on cytosines preceding guanines, called CpG dinucleotides [12]. DNA methylation patterns are established during early development and are maintained in differentiated tissue by DNA methyltransferases [13]. Changes in DNA methylation are a potential mechanism by which the expression of a gene may be regulated [12]. For example, it is generally accepted that gene expression is often reduced when DNA methylation is present at a promoter or untranslated region of a gene [14–16]. There have been a number of studies that have focused on the epigenetic basis of obesity [17, 18]. However, the majority of the DNA methylation studies performed to date have either used a candidate gene approach or the array based technology that probes 450K methylation sites simultaneously. Therefore, our study is unique in that we performed reduced representation bisulfite sequencing (RBBS), which has the ability to capture millions of methylation sites in the human genome. Moreover, we performed transcriptomic analyses, which Page 2 of 12 allowed us to measure global messenger RNA (mRNA) expression levels in genes altered in people with obesity. Furthermore, we combined epigenetic and transcriptomic analyses to identify associations between the datasets. Based on our previous findings in skeletal muscle, we hypothesize that there will be alterations in the methylation of genes involved in mitochondrial function, inflammation, and (...truncated)